Summary A major signaling paradigm for modulation of hormonal and other extracellular stimuli is the use heterotrimeric G proteins by cell surface receptors. Besides direct regulation of intracellular enzymes that produce second messengers, these receptor/G protein pathways influence the action of several members of the Ras superfamily of monomeric GTPases. Members of this family, such as Ras and Rho proteins, regulate cellular growth, differentiation, shape and adhesion. P115-RhoGEF and its homologs can directly modulate the exchange of GTP on RhoA when activated by the heterotrimeric G13 protein. In turn, the RGS (regulator of G protein signaling) domain of p115-RhoGEF can stimulate the GTPase activity of G13 and thus its inactivation. This proposal continues examination of the regulatory mechanisms among components of these pathways in vitro and will attempt to clearly define their physiological roles in cellular regulation. Proposed studies include attempts to determine structures of regulatory complexes using both classical crystallography and small-angle x-ray scattering (SAXS). Mutational analysis and reconstitution of coupled reactions in vitro and in cells will examine proposed mechanisms, especially the role of GTPase stimulation for either facilitation or Hormone Receptor G13 RhoGEF RhoA Functions inhibition of hormonal signaling and translocation to cellular membranes. Fluorescent sensors will be developed and used to assess the actual activity and location of G13 in vivo and relate this to putative functions including activation of Rho, modulation of cAMP, and cellular motility. Fluorescence, mutagenesis, exogenous expression and gene-silencing techniques are proposed to determine roles for signaling complexes and specificity of signaling in these pathways with a focus on regulation via receptors for lysophosphatidic acid and sphingosine-1-phosphate. Mutations in p115-RhoGEF and its homologues are known to be oncogenic and G13 is required for development. Progress will increase our understanding of these key pathways in regulating growth, differentiation and cell motility. This will help to better understand the regulation imparted by a variety of hormones and the contribution of these proteins to cellular dysfunction and disease.